The goals of this project are to understand recombinational DNA repair, particularly the mechanisms of the central recombinases involved in this process and their regulation. Homologous genetic recombination is at once (a) a key DNA repair process, (b) one of the important cancer avoidance pathways in higher eukaryotes, and (c) one of the primary paths to the productive alteration/engineering of cellular genomes. The competing proposal is focused on the bacterial RecA protein and proteins that regulate or augment RecA function. We will also explore specific applications of these proteins in biotechnology. There are four specific aims. Aim 1 is focused on RecA itself. One key initiative seeks to generate RecA variants with enhanced functionality. Aim 2 is concerned with RecA regulators. These include the RecFOR proteins that load RecA onto ssDNA, the PsiB protein that binds to free RecA protein and inhibits its binding to DNA, the DprA protein that also helps load RecA onto DNA and enhances DNA transformation in vivo, and the UvrD helicase that is responsible for displacing RecA filaments from the DNA when they are no longer needed. As part of aim 2, we will also explore the function of MgsA, an AAA+ ATPase involved in genome maintenance that is highly conserved from bacteria to humans. In Aim 3, we will explore the function of a novel RecA-dependent nuclease called Ref, discovered during the last grant period. Ref, encoded by P1, can be used as a kind of universal restriction enzyme. Deployed with RecA, Ref will efficiently introduce targeted double strand breaks at any chosen DNA location in an oligonucleotide- directed fashion. Finally, Aim 4 will continue our efforts to elucidate the role of RecA protein in the function of the mutagenic DNA polymerase V. Practical applications include the use of the Ref nuclease for targeted DNA cleavage, and the use of RecA-mediated double strand break repair for metagenomics, filling gaps in large genomic sequencing projects, and the enhancement of forensic DNA analysis. Ref may eventually be utilized in the genomic engineering of mammalian DNA, in the service of human gene therapy or the generation of mouse gene knockouts.